SUMMARY Muscle contraction and cell motility are mediated by the interaction of myosin motors with actin filaments. The free energy from ATP hydrolysis is used to produce conformational changes in the actomyosin that produce force and movement. These conformational changes are associated with actomyosin interaction that promote successive release of phosphate (Pi) and ADP. Despite a detailed knowledge of the kinetics of actomyosin ATP hydrolysis in which actin accelerates the dissociation of Pi and ADP from myosin, our understanding of the molecular mechanism(s) by which the free energy of ATP hydrolysis is coupled to the production of work and movement by myosin motors remains elusive. High resolution x-ray crystal structures of the myosin head alone are available in various biochemical states, but due to resistance of filamentous actin (F-actin) to crystallization the only tool to reveal molecular details of force generation by myosin motors remains cryo electron microscopy (cryo-EM). Recent cryo-EM structures of nucleotide free (rigor) actomyosin and actomyosin-ADP provided a mechanistic model of ~20 percent of the crossbridge movement that occurs at the end of the myosin work cycle, but a high resolution structure of the actomyosin-ADP-Pi intermediate that precedes the remaining 80% of the powerstroke associated with Pi dissociation is still unavailable. Studies of the structure of the prepowerstoke actomyosin-ADP-Pi have been unsuccessful because myosin-ADP-Pi is weakly bound to actin (Kd ~30 uM) and the Pi rapidly dissociates (< 5 ms for myosinVa) to form actomyosin- ADP. Because 80% of the crossbridge movement is associated with the dissociation of phosphate from actomyosin-ADP-PI, a detailed structure of actomyosin-ADP-Pi is crucial for understanding what is arguably the most important step of the actomyosin ATP hydrolysis mechanism. The ultimate aim of this application is to utilize a unique and sophisticated apparatus for millisecond time resolved cryo-EM combined with a state of the art electron microscope equipped with direct detector and new image processing techniques to produce high resolution 3D reconstructions of the prepower stroke actomyosin-ADP-Pi complex. In addition to these technical advances we will use site directed mutagenesis to produce myosin-ADP-Pi complexes that have 10- fold increased affinity for actin and 10-fold reduced rates of Pi dissociation. A high resolution structure of the prepowerstroke actomyosin-ADP-Pi complex will enable us to better understand how myosin motors function in muscle and non-muscle cells and open new avenues for treatment of myosin involved diseases such as cardiomyopathies and cancer metastasis.